Virtual separation of bound and free label in a ligand assay for performing immunoassays of biological fluids, including whole blood

ABSTRACT

Detection and characterization of immunologically detected substances are performed electronically on human and animal biological fluids such as whole blood, serum, plasma, urine, milk, pleural and peritoneal fluids, and semen, which fluids are contained in a thin chamber forming a quiescent fluid sample, which chamber has at least two parallel planar walls, at least one of which is transparent.

This application is a continuation of U.S. patent application Ser. No.13/204,416 filed Aug. 5, 2011, which is a continuation of U.S. Pat. No.7,995,194 filed Apr. 2, 2009, which claims the benefit of U.S.Provisional Applications Nos. 61/041,784, filed Apr. 2, 2008;61/041,791, filed Apr. 2, 2008; 61/041,790, filed Apr. 2, 2008;61/041,794, filed Apr. 2, 2008; 61/041,797, filed Apr. 2, 2008; and61/043,571, filed Apr. 9, 2008.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to the virtual detection, quantization andcharacterization of immunologically detected substances electronicallyin human and animal biological fluids such as whole blood, serum,plasma, urine, milk, pleural and peritoneal fluids, and semen, whichdetection, quantification and characterization is performed in a thinchamber on a quiescent fluid sample, said chamber having at least twoparallel planar walls, at least one of which is transparent.

2. Background Information

This invention relates to the improvement in the performance of allimmunoassays that presently involve the physical separation of boundfrom free analyte by, instead, performing a virtual separation of boundand free optically detected label in a ligand assay, wherein the labelis preferably a fluorescent label although any optically detectable andquantifiable label will suffice. The chambers for use in this assay andthe instruments for measuring the analytes in these chambers aredescribed in the following issued U.S. Pat. Nos. 6,929,953 issued to S.C. Wardlaw; 6,869,570 issued to S. C. Wardlaw; 6,866,823 issued to S. C.Wardlaw; and U.S. Patent Application Publication No. US 2007/0087442, toS. C. Wardlaw, published Apr. 19, 2007.

Physical separation of bound from free analytes have, in the prior art,been accomplished by multiple means including but not limited to,adsorption of the free label by charcoal or talc, magnetic separation ofbeads containing either the bound or unbound analyte, adsorption of thebound labeled analyte by the container such as antibodies coupled to thewall of a test tube and the use of second precipitation antibodiesdirected against the analyte binding antibody followed by centrifugationas well as the methods described in the above noted patents andpublications.

Some of the types of prior art physical separation of bound targetanalyte assays are described in the following U.S. Pat. Nos. 5,834,217;5,776,710; 5,759,794; 5,635,362; 5,593,848; 5,342,790; 5,460,979;5,480,778; and 5,360,719, all issued to R. A. Levine et al. In theaforementioned patents, the separation of bound from free analyte isperformed by centrifugation, or other physical methods, such asdecanting, filtration, or the like.

The prior art also describes a type of immunoassay, which is called a“homogeneous immunoassay”. Homogeneous immunoassays do not require thephysical separation of bound from non-bound, or free, analyte. The“separation of the bound from free” is accomplished by utilizing thesteric interference of an enzyme by the relatively large antibody andquantifying the colored or fluorescent products of the enzymatic action.Additional methods of homogeneous assays utilize the fluorescentquenching of fluorophores to distinguish bound from free analyte. Whilethese methods greatly simplify the performance of immunoassays, they aregenerally useful only for high concentrations of analytes with lowmolecular weight since the large molecular weight of target analytessuch as proteins (e.g., insulin), growth hormones, and the like willalso interfere with the enzyme and may affect quenching Additionallyimmunoassays of this homogeneous type typically do not have the highsensitivity of standard immunoassays.

It would be highly desirable to provide a ligand assay of a targetanalyte wherein the quantification of the target analyte is a virtualone which can be performed electronically thereby having the advantagesof a homogeneous immunoassay while maintaining the sensitivity ofstandard immunoassays, as well as the ability to have large size targetanalytes such as hormones like insulin, growth hormone and the like.

SUMMARY OF THE INVENTION

Immunoassays are used to analyze a wide range of analytes, such ashormones in blood, etc. They work by the general technique of finding aspecific binder which specifically binds to the target analyte beingmeasured. A binder is referred to herein as a ligand. Ligands aredefined herein as including, but not limited to those antibodies,lectins, aptimers, or naturally occurring substances, that are operativeto bind a target analyte. The sample to be measured is admixed with theligand which is specific to the target analyte, and a labeled version ofthe analyte to be measured. As this mixture is incubated, the labeledand unlabeled target analyte molecules compete for binding sites on theligand. After a suitable period, the ligand is removed by any number ofways, and the label bound to the ligand is compared to the label whichis unbound and remains free in the mixture. This bound/free ratiorelates to the concentration of the target analyte originally in thesample, although either the bound or free label can give the sameinformation. The use of the ratio allows the quality control checkwherein the total of bound plus free is relatively constant if thevolume is constant. This quality control may also be employed in thepractice of this invention.

According to an aspect of the present invention, a method for assaying abiological fluid sample for a target analyte material that may be in thefluid sample is provided. The method utilizes a virtual separation offree and bound target analyte disposed within the fluid sample involvingelectronic scanning of the sample. The method involves placing the fluidsample in a test chamber having a predetermined and fixed height so asto produce a thin layer of the fluid sample in the chamber. At least onewall of the chamber is transparent, usually the top wall, so that thesample can be observed in the chamber. In certain cases both the top andbottom walls of the chamber are transparent. The height of the chamber(e.g., typically 1 μ to 200 μ) can vary according to the application athand. For example, when anticoagulated whole blood is being analyzed, achamber height of 6 μ is advantageous because it creates a monolayer ofred blood cells and interspersed plasma lacunae within the blood sample

The height of the fixed structure or ligand-coated bead optimally shouldbe no less than one tenth of that of the chamber and ideally approachingthe height of the chamber. The reason for this is that if the totalamount of label (e.g., fluorophore) present in the free state,surrounding the particle or structure to which the label is bound, ismuch greater than the amount of the lowest amount of bound label to bedetected, the ability to accurately determine the amount of label boundto the bead or structure is diminished due to the influence of signal tonoise ratios. Mathematically there is no limit to the height of thechamber but practical limits due to signal to noise of the detectedlabel require a thin chamber and structures occupying at least tenpercent of the volume of a cylinder drawn around the periphery of thestructure and extending from the base to the tip of the chamber foroptimal function. In examples where the ligand is adherent to thechamber top or bottom rather than a structure or bead, the above ratiosapply, but the assay optimally should be formulated so that thecylindrical volume above the bound ligand area contains not more thanten times the lowest amount bound to the ligand area that is desired tobe detected. This constraint can be diminished by making the chamber asthin as possible or by altering the stoichiometry of the reaction.

The method of this invention can be used to test for drug allergies orallergen sensitivities in patients at the point of care. Drug allergiesand allergen sensitivities are a common and important problem. It isexpensive to the patient and society. Treating a penicillin allergicpatient with a penicillin class drug, for example, can cause death orserious reactions. Penicillin is used in this discussion as arepresentative drug and because it is the drug type that is the mostcommon cause of severe allergic reactions. The present invention is notlimited to testing for penicillin allergies, and can be used to test forsensitivity to other drugs (e.g., antibiotics, muscle relaxants,anesthetics, etc.) and allergens.

Penicillin is an inexpensive, effective and generally non-toxic drug.Patients who think they have a penicillin allergy, can be treated withanother less microbial-targeted drug in view of the perceived allergy.Such replacement drugs may cause serious side effects in patients,however, and incur enormous costs to the health care system, since newermedications can be hundreds or thousands of times more expensive thenpenicillin drugs. Equally importantly are the costs to societyassociated with the increased development of drug resistant bacteria,viruses, or other infectious agents that occurs when broader spectrumdrugs are used instead of drugs more focused on the target organism. Itis, therefore, important to individual patients, healthcare providers,and society, to determine the presence or absence of drug allergies orallergen sensitivities by methods in addition to the history given bythe patient. One goal of this invention is to detect the presence orabsence of drug allergies and/or allegen sensitivities in a sample of apatient's whole blood or plasma.

It is well documented that many patients who claim to be allergic topenicillin are not allergic and similarly some patients who think thatthey are not allergic may have developed an allergy since their lastexposure. There are many reports that about 80% of individuals whobelieve they are allergic to penicillin will in fact tolerate penicillinuse, so for these patients the constraints on antibiotic choice,potentially resulting in less effective, more toxic and more expensivetreatment, are unnecessary.

Nowhere is the need for the ability to detect drug allergy more neededthan at point of care encounters with the patient. Physicians about toprescribe a medication in their office, the emergency room or hospitaldo not have the luxury of waiting many hours or a day for the test to beperformed either in vitro, or by skin tests. Skin testing mayadditionally expose the patient to risk of reaction to the testingsubstance and has the theoretical possibility of inducing allergy orincreasing it by an anamnestic response. In vitro tests at present arecomplex, time consuming to perform and yield information to thephysician long after it would be most useful. Additionally theallergenic nature of many drugs, including penicillin type drugs, may bedue to more than one epitope and accurate testing would require testingfor all common epitopes which may be the cause of the allergic response.RAST testing, well described in the literature, is generally performedon a limited number of test allergens and their epitopes.

It is generally agreed that IgE mediated immune response is the cause ofmost severe allergic reaction including anaphylaxis, hives, intestinalswelling with diarrhea and respiratory obstruction due to swelling ofairways. It is suggested by some experts that other immunoglobulinclasses may also contribute to the allergic response to drugs butgenerally the allergic response to IgG and IgM mediated drug allergiesis not life threatening and more likely to be a rash.

An advantage of the present invention is that it provides a means toperform, optimally at the point of care, a determination of the presenceof IgE or any other immunoglobulin which has an affinity for one or moredrugs that are or may be indicated for use in a given situation.

The label of choice is the use of a fluorophore that is easily detectedand attached to the ligand. The present invention is not limited tousing fluorometric labels, however. More than one color fluorophore maybe used if it is desired to check for the presence or absence of morethan one class of immunoglobulin that may become attached to the beadsin the same chamber. Beads without the attached antigen are used ascontrols. The control beads can be chemically and geometrically similarto the coated beads, differing only in color or other means enablingtheir detection (e.g., fluorescence or combinations of fluorescence dyesincorporated into their structure). The control beads provide a controlso that the detection, for example of significant fluorescent signalfrom the fluorescent labeled antibody directed against the IgE that isattached to the beads containing a determinant (epitope) of the drugbeing tested as a potential allergen, may be compared to the signal thatis present of similar beads not coated or bound to the epitope. Thus,nonspecific binding is controlled and will not result in a falsepositive.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side view of a ligand-bearing surface which may beused to perform an immunoassay on a blood or other sample in accordancewith the prior art.

FIG. 1( a) is a view similar to FIG. 1 but showing the surface after thesample has been washed away therefrom in accordance with the prior art.

FIG. 2 is a schematic side view similar to FIG. 1, but showing aligand-bearing surface which may be used to perform a sandwichimmunoassay on a blood or other sample in accordance with the prior art.

FIG. 2( a) is a view similar to FIG. 2 but showing the sample after asecond label has been added to the sample in accordance with the priorart.

FIG. 2( b) is a view similar to FIG. 2( a) but showing the surface afterthe sample has been washed away therefrom in accordance with the priorart.

FIG. 3 is a plan view of a first embodiment of a sampling chamber formedin accordance with this invention which contains an anticoagulated wholeblood sample to which blood sample ligand-bearing analyte-capturingparticles have been added.

FIG. 4 is a side sectional view of a portion of the sampling chamber ofFIG. 3 which contains one of the ligand-coated target analyte-capturingparticles.

FIG. 5 is a plan view of a test chamber like that shown in FIG. 4,showing an area of the sample containing one of the ligand-coated targetanalyte-capturing particles and also showing another area of the samplewhich does not contain one of the ligand-coated target analyte-capturingparticles but only contains the free labeled target analyte in the bloodsample.

FIG. 6 is a fragmented cross-sectional view of a partially ligand coatedtarget analyte capturing surface wherein portions of the surface arecoated with ligands and other portions of the surface are not.

FIG. 7 is a sectional schematic view of a closed chamber having a topsurface such as that shown in FIG. 6.

FIG. 8 is a plan view of the surface shown in FIG. 6.

FIG. 9 is a trace of the emissions from the ligand bands on the capturesurface shown in FIGS. 6 and 8.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, FIGS. 1 and 1( a) illustrate a prior artcompetitive immunoassay (also referred to as an “equilibrium assay”)which is commonly used for analytes of low molecular weight, such as thethyroid hormone, thyroxin, where the numeral 1 denotes a surface towhich a ligand 2, which is specific to the target analyte, is attachedby any number of means well-known to the art. Surface 1 may be atransparent wall of a glass or plastic tube or a particle. A solution 3contains a mixture of the unlabeled target analyte 4 (the unknown) and alabeled target analyte 5. After a period of time, which may be fromminutes to hours, depending upon the target and the label, the labeledtarget analyte 5 and the unlabeled target analyte 4 will be in anequilibrium with each other, wherein many, but generally not all, of theligand sites 2 will be occupied with either a labeled target analyte 5or unlabeled target analyte 4. At this point (FIG. 1 a), the mixture 3is separated from the ligand-bearing surface 1 in a manner thatpreserves the labeled target analytes 5 which are bound to the ligand 2.The labeled target analytes 5 bound to the surface 1 are then measured(see FIG. 1 a), and the free labeled target analytes may also bemeasured or may be calculated as: Total=Free+Bound, or Bound=Total−Free.The bound to free target analyte ratio is inversely related to the totaltarget analyte amount in the sample.

FIG. 2-FIG. 2(b) show a ligand assay often referred to as a “sandwich”assay, where two separate ligands are utilized. Surface 1 has ligand 2bound (“bound ligand”) thereto in a similar manner described above, andthe sample containing the target analyte 4 is introduced into thesolution 3 and incubated with the surface 1. Either immediately, orafter a suitable period of time, a separate labeled ligand 6 isintroduced into the solution, which labeled ligand 6 binds to a site onthe target analyte 4 which is different than bound ligand 2 (FIG. 2 a).This, in effect, creates a “sandwich”, containing the target analyte 4in the center. The free labeled ligand 6 is then washed off the surface1 to leave the surface 1 covered with labeled sites (FIG. 2 b). Thelabeled target analytes 4 bound to the surface 1 are then quantified,and the signal therefore is directly proportional to the amount of thetarget analyte 4 in the original sample. It is generally recognized thatthe sandwich assay is more precise and somewhat more accurate, but itcan only be applied to target analyte molecules which have at least twodifferent sites to which ligands can be bound.

In either of the above assays, the separation of the bound label fromthe free label is recognized as one of the challenging aspects of theprocedure, and often requires one or more mechanically complex steps,such as centrifuging, decanting, washing, etc. As a result,instrumentation to automate these tests has been relatively complex,requiring multiple operations.

Aspects of the present invention, in contrast, provide a means of“virtual separation”, wherein the bound and free label are notphysically separated, but rather separated by a combination of test cellconfiguration and mathematical manipulation of the signals fromdifferent regions in the test cell. As a result, simplified automatedligand assay methods and apparatus can be performed.

According to aspects of the present invention, immunoassays or ligandassays are performed where the binder is a ligand or other a substancehaving a high affinity for the target analyte.

Assays according to the present invention can be performed, for example,using the sample containers and imaging instrument systems described inthe U.S. Patent Publication Nos. 2007/0243117 and 2007/0087442 and U.S.Pat. No. 6,866,823, all of which are hereby incorporated by reference intheir entirety. The present assays are not limited to these chambers andimaging devices, however.

The term “immunoassay” as used in this disclosure and claims shall meanboth antibody-based binding agents and non antibody-based bindingagents. Examples of the latter include, but are not limited to,intrinsic factors for binding vitamin B12, and avidin for bindingbiotin-labeled targets or vice versa.

Under aspects of the present invention, a well-defined and physicallycircumscribed surface is provided to which the ligand is attached, andthen the signal from the label bound to that surface is mathematicallydistinguished from that of any surrounding free label that may reside insolution. There are two general cases which are described as follows.

FIG. 3 is a plan view of a section of a specimen chamber assembly 40,which chamber assembly 40 contains an anticoagulated whole blood sample.The chamber assembly 40 includes upper and lower walls 7 (see FIG. 4),at least one of which is transparent. Preferably, both of the walls 7are transparent. The chamber assembly 40 includes spacer members 42 (seeFIG. 3) which are randomly located inside of the chamber assembly 40.The spacer members 42 are preferably spherical and determine and controlthe height of the chamber assembly 40. In the case of assaying ananticoagulated whole blood sample, spacer members 42 having a diameterof about 6 μ work particularly well. The blood sample which is containedin the chamber assembly 40 will include individual red blood cells 44and agglomerations of red blood cells 46. The blood sample also includesclear plasma lacunae areas 48 which do not contain any formed bloodcomponents. Finally, the blood sample also includes a plurality ofligand-coated target analyte-capturing particles 8 which are preferablyin the form of spheres. The target analyte-capturing particles 8 arerandomly distributed throughout the blood sample, and may be about 3 μ-4μ in diameter for a blood sample analysis, so that they can be easilydetected in the blood sample.

FIG. 4 shows the structure of the chamber assembly 40 of FIG. 3. Thechamber assembly 40 is bounded by top and bottom wall 7, at least one ofwhich must be transparent. Within the chamber is a particle 8, whosesurface is covered with a ligand 9. The particle 8 may be any shape aslong as its volume can be determined, but it is preferably a sphere. Theparticle 8 may be of any material to which a ligand can be attached,such as glass, polystyrene, or the like. The particles are not limitedto any particular diameter (e.g., 2 μ-100 μ), and the diameter can varydepending on the fluid being assayed and the height of the chamber beingused. The distance between the walls 7 is typically not less than thediameter of the particle 8, but the upper distance limit will dependupon the nature of the particle 8.

A mixture 10 contains both a target analyte 11 and a labeled targetanalyte 12 in a manner similar to that described in connection with FIG.1 above. After a suitable period of incubation, the signals from thebound and free target analyte are processed.

FIG. 5 is a top view of a test chamber assembly like that shown in FIG.4, showing an undefined expanse 13 of the mixture 10. Within thisexpanse, the total signal from the label 12 is collected over a definedarea 14, which area is not limited to any particular shape. The means ofcollection can be a fluorescence scanner, in the case of a fluorescentlabel, or a radio nucleotide scanner, in the case of a radio label. Thearea is chosen so that it includes at least one particle 8, with a knownor measurable diameter. An adjacent defined area 16, not containing aparticle, is also measured. The signal from area 16 represents that fromthe unbound label, since there are no binding sites in that location.The signal from area 14, however, has a signal from both the bound andthe free label. The influences of each can be determined in a number ofways. If the particle is spherical, which is a preferred shape, itsvolume (Vp) can be calculated from its diameter, which can be measuredwith the same optical system that collects the signal from the label.The volume of the defined areas 14 (V14) and 16 (V16) can be readilycalculated from their width and the chamber depth. Assuming that thechamber volumes associated with defined areas 14 and 16 are identical,the signal from the free label is equal to that of the signal from area16 (S16). This means, that in the absence of signal from the particle(the bound label), the signal from area 14 (Sf) should be:Sf=S16−(V14−Vp). Any signal in excess of this amount is from the boundlabel (Sb): Sb=S14−Sf. If the volume of the particle is de minimuscompared to the volume within the area 14, then the volume correction isnot necessary. What is determined is the average label signal intensityper pixel (or collective group of pixels) of the scans. The term pixelas used in this application may include the meaning of one or moreadjacent pixels.

In a second, and most preferred embodiment, ligands are attached to atleast one surface of the chamber itself. FIG. 6 shows an (upper)transparent chamber surface 17, which may be glass or plastic, such asacrylic or polystyrene, to which a uniform coating of the ligands hasbeen attached by any number of means well known to the art. After theuniform coating is formed, ligand are selectively removed from one ormore regions 18, either by mechanical or chemical means, or by laserablation, consequently leaving active ligands in adjacent regions 19.

FIG. 7 shows this surface 17 as part of a thin chamber containingmixture 20, comprising unlabeled target analyte 21 and labeled analyte22. The chamber is preferably less than about 1 mm in height, and ismost preferably less than 200 μ (e.g., in a range of 1 to 200 μ). Asbefore, after a suitable period of time, the labeled and unlabeledanalyte will reach equilibrium with the ligand, leaving a portion of thelabeled analyte 23 bound to the surface, but only in the region wherethe ligand remains. In the case of a fluorescent label, the chambersurface 17 is illuminated with light source 24 of the appropriatewavelength to excite fluorescence in the label. Lens 25 collects thefluorescent emissions, which are filtered by optical filter 26 andprojected onto an image dissection device 27, which may be a chargecouple device (CCD), complimentary metal oxide semiconductor (CMOS), orthe like. Alternatively, the light source may be a laser which focuses atiny, moving spot onto the chamber, and the light collecting device 27would be, in that case, a simple phototube or photomultiplier.

The net result of either process is shown in FIG. 8, which is aschematic top view of the chamber 28, where the active ligand 29 andablated ligand 30 appear as a series of vertical stripes. The scan linesfrom the apparatus of FIG. 7 are represented by the lines a-a. FIG. 9 isa representation of the waveform taken across the scan lines a-a, wherethe peaks 31 are the signal from the active ligand, and the valleys 32are from the inactive areas. Thus, the bound label concentration isrepresented by the distance from the peaks to the valleys, and theheight of the valleys represents the free label. The active areas andinactive areas are not limited to any particular geometry.

In some embodiments, a chamber wall 17 can be used that is sufficientlyflexible that it can be locally elastically deformed by subjecting it toa relatively small point load. The elastic nature of the chamber wall 17allows a unique option to capture very weak “bound” signals. If thechamber wall 17 is compressed, such as by a small stylus just out of theimaged area, the free label 22 is expelled laterally from the localfield of view, and thus its signal is markedly reduced. With this“background” signal reduced, very weak signals from bound label 23 canbe detected.

Multiple analytes could be measured simultaneously if the labelsfluoresce at different wavelengths, or if the ligand for analyte 1 wereat a different physical location in the chamber from the ligand foranalyte 2.

An example of a method according to the present invention methodincludes performing an assay to determine whether a patient may beallergic to one or more drugs (e.g., antibiotics, including penicillin,etc.) or allergens. The assay is performed using a cartridge that has ananalysis chamber containing a large number (e.g., thousands) ofantibiotic epitope coated beads and uncoated control beads. For thoseanalyses directed toward more than one antibiotic epitope, eachparticular antibiotic epitope is matched with a particular type of beadfor identification purposes. The groups of beads associated withdifferent epitopes can be distinguished from one another usingcharacteristics such as a bead color, size, shape, etc; e.g., epitope Ais coated on white beads, epitope B is coated by red beads, etc. A smallamount of sample (e.g., 0.5 to 5 micro liters) of capillary or venousanticoagulated whole blood is deposited in the chamber (e.g., drawn intothe chamber by capillary action) and upon closing the chamber the bloodis directed into an area within the chamber containing the beads. Afterincubation for a first period of time (e.g., minutes to an hour)immunoglobulin present within the sample binds to those beads coatedwith a drug (or allergen) to which the immunoglobulin molecule has aspecific affinity. Different immunoglobulin molecules present within thesample may have different affinities specific to different drugs (orallergens). The combined beads and blood sample is further mixed withone or more labeled antibodies directed against the immunoglobulin beingtested (e.g., Immunoglobulin E (“IgE”), etc.) and allowed to incubatefor a second period of time (e.g., seconds to minutes). A fluorophoremay be tagged to the antibodies directed against the immunoglobulinbeing tested to create the “labeled antibody”. The sample is thendirected into the analysis chamber of the type described above. Theactual times needed for incubation for the two steps can be empiricallydetermined and will likely depend upon the avidity and concentration ofthe antibodies present. The sample disposed within the chamber isanalyzed by collecting the signal from the labeled antibodies both freeand bound in one or more of the manners described above. If the assayinvolves the determination of allergy susceptibility of more than onedrug, or sensitivity to more than on allergen, the analysis will includedistinguishing the bound labeled antibodies as a function of thedifferent types of coated beads as well. The bound label representsthose labeled antibodies that are bound to the immunoglobulin beingtested, which immunoglobulin is bound to the particle coated with thedrug (or allergen) with which the particular immunoglobulin particle hasa specific affinity. The amount of label bound on a particle may becalculated by measuring the total signal of the imaged particle andsubtracting the surrounding free signal in the immediate areasurrounding the particle that is included in the image. Ratios of theamount of label on a given class of coated beads can be calculated bymeasuring labeled coated and uncoated beads of the same type. Thus, thedetermination of whether a sample contains immunoglobulin moleculeshaving an affinity for a given drug (or allergen) can be performed bypracticing the present invention. In addition, simultaneous detection ofan allergy to more than one drug (or sensitivity to more than oneallergen) can be performed under the present invention using differenttypes of detectable beads or particles, with each type coated with adifferent drug (or allergen). A single type particle (or bead) may beused as a control particle for all the drug allergy (allergensensitivity) tests if the particle is the same in size and compositionas the coated particles. If necessary, more than one type of controlparticle may be used with the size of the control particle matching thesize of the drug or allergen coated particles to which it is beingcompared.

In some embodiments, after the second incubation (the one containing thelabeled ligand) the method includes the step of adding a liquidcontaining no label to the sample containing unbound label disposedwithin the chamber, thereby leaving primarily the label attached to theimmobilized beads or structures. The virtual separation of bound fromfree is subsequently performed as previously stated but the removal ofthe liquid containing the label can serve to increase sensitivity of theassay at the expense of complexity. Since the total capacity of thechamber and the amount of liquid in the chamber is in the range of lessthan one to several micro liters, the addition of a label-free fluid tothe chamber in a substantial volume (e.g., tens of micro liters) willremove much of the fluid containing label and the remaining free labelsignal will be removed by the utilization of the virtual separation ofbound from free process.

The above described methodology provides a novel and desirable techniquefor determining the amount of bound and free labeled target analytewithin areas of a chamber that contain ligands or are free of ligandsspecific to that target analyte within a sample, and thereby providesqualitative and quantitative information relative to the sample. In someinstances, qualitative information such as knowing whether the targetanalyte is present or absent in the sample is sufficient information forthe analysis at hand. An example of such an instance is thedetermination of whether a specimen has specific IgE directed against agiven drug, when the absence of such IgE is the normal state. If morequantitative information is desired (e.g., the concentration of thetarget analyte in the sample), the obtained bound/free information maybe used with a standard curve, which curve is empirically derived forthe particular target analyte and sample being considered, to determinethe quantitative information; e.g., the amount of target analyte withinthe sample. Standard curves operable to be used with all types ofimmunoassays are known and the present invention is not limited to anyparticular standard curve. Sample curves may be performed prior to orconcurrently with the assay and the results stored on the instrumentperforming the analysis.

Although the invention has been shown and described with respect tospecific detailed embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and detail thereof maybe made without departing from the spirit and the scope of theinvention.

What is claimed is:
 1. An apparatus for performing a target analyteimmunoassay of a fluid sample including a detectable label, which labelis operative to bind to the target analyte to produce labeled targetanalyte, which labeled target analyte is detectable in the sample, theapparatus comprising: a sample chamber having at least one first surfacearea that has target analyte specific ligands attached to it, and atleast one second surface area that is free of target analyte-specificligands operative to selectively bind target analyte present in thesample to the second surface area; and an imaging device adapted tooptically scan the at least one first area to determine a label signalintensity value per unit distribution of the labeled target analyte inthe at least one first area, and adapted to optically scan the at leastone second area to determine a label signal intensity value per unitdistribution of the labeled target analyte in the at least one secondarea, and adapted to determine at least one of an amount of labeledtarget analyte in the at least one first area, an amount of labeledtarget analyte in the at least one second area, and a ratio of labeledtarget.
 2. A method for performing a target analyte immunoassay of afluid sample disposed within a chamber comprising the steps of:providing a detectable label within the sample, which label is operativeto bind to the target analyte to produce labeled target analyte;providing at least one first surface area within the chamber which firstsurface area has target analyte specific ligands attached to it, whichtarget analyte-specific ligands are operative to selectively bind targetanalyte present in the sample to the first surface area; providing atleast one second surface area in the chamber which is free of targetanalyte-specific ligands; optically scanning the at least one first areato determine a label signal intensity value per unit distribution of thelabeled target analyte in the at least one first area; opticallyscanning the at least one second area to determine a label signalintensity value per unit distribution of the labeled target analyte inthe at least one second area; and determining at least one of an amountof labeled target analyte in the at least one first area, an amount oflabeled target analyte in the at least one second area, and a ratio oflabeled target.
 3. A sample chamber for performing a target analyteimmunoassay of a fluid sample that includes a detectable label, whichlabel is operative to bind to the target analyte to produce labeledtarget analyte, the sample chamber comprising: at least one firstsurface area, which first surface area has target analyte specificligands attached to it, which target analyte-specific ligands areoperative to selectively bind target analyte present in the sample tothe first surface area; and at least one second surface area in thechamber which is free of target analyte-specific ligands.